Composite biomaterials

ABSTRACT

This invention provides novel composite biomaterials having excellent bioadaptability and bone inductivity and a process for producing the same. The composite biomaterials comprise hydroxyapatite, collagen, and alginate and have microporous structures in which the c-axis of the hydroxyapatite is oriented along the collagen fibers.

TECHNICAL FIELD

The present invention relates to composite biomaterials comprisinghydroxyapatite, collagen, and alginate and a process for producing thesame. Particularly, the present invention relates to novel compositebiomaterials having mechanical properties similar to those of naturalbones, excellent bioadaptability, and excellent bone-conductivity and aprocess for producing the same.

BACKGROUND ART

In the field of regenerative medicine, a variety of artificialbiomaterials that can be used as substituents to damaged tissues ororgans have been recently developed. Implants such as artificial bonesor artificial bone fillers are used particularly for treatment of bonedefects. Such implants, however, are required to have bioadaptabilityand bone inductivity in addition to mechanical properties similar tothose of natural bones. That is, implants needs to be gradually resorbedafter implantation in the body, involved into the bone regenerationcycle, and then substituted for the bones of the subject.

Bones of vertebrates are composed of hydroxyapatite and collagen. Theyforms a specific nanocomposite structure in natural bones characterizedin that the c-axis of hydroxyapatite is oriented along the collagenfibers, and this structure imparts bone-specific mechanical properties.Composite biomaterials of hydroxyapatite and collagen having structuresand compositions similar to those of natural bones are described in, forexample, JP Patent Publication (Kokai) Nos. 7-101708 A (1995) and11-199209 A (1999), and bone inductivity thereof has been observed tosome extent.

Alginic acid is a polysaccharide contained in seaweed, which has beenheretofore employed in a hemostatic drug or wound dressing. Concerningartificial bones, development of bone fillers comprising α-TCP incombination with alginate has been reported (Nagata, Shika Zairyou(Dental materials), vol. 16, No. 6, 1997, pp. 479-491). Moreover,alginic acid has been recently reported to help a repair of bones and/orcartilages (e.g., Fragonas et al., Biomaterials 21, 2000, pp. 795-801).Application of alginic acid to a composite of hydroxyapatite andcollagen, however, had not yet been attempted. In particular, homogenousincorporation of alginic acid into the composite while maintaining itsspecific nanocomposite structure involves some difficulties.

DISCLOSURE OF THE INVENTION

An object of the present invention is to provide novel compositebiomaterials having excellent bioadaptability and bone inductivity inwhich alginate is homogenously distributed in a composite ofhydroxyapatite and collagen having microporous structures similar tothose of natural bones and a process for producing the same.

The present inventors have conducted concentrated studies in order toattain the above object. As a result, they have succeeded in obtaining acomposite in which alginate has been homogenously incorporated by addingalginate to a microporous composite of hydroxyapatite and collagen in agiven step and curing the mixture. They have found that this compositehad excellent bioadaptability and bone inductivity. This has led to thecompletion of the present invention.

More specifically, the present invention provides the following (1) to(10).

(1) Composite biomaterials, which comprise hydroxyapatite, collagen, andalginate and have a microporous structure in which the c-axis ofhydroxyapatite is oriented along the collagen fibers.

(2) The composite biomaterials according to (1), wherein an alginatecontent is 1 to 30% by mass, relative to the total amount ofhydroxyapatite and collagen.

(3) The composite biomaterials according to (2), wherein an alginatecontent is 5 to 20% by mass, relative to the total amount ofhydroxyapatite and collagen.

(4) The composite biomaterials according to any one of (1) to (3),wherein the ratio of hydroxyapatite with collagen is between 60:40 and90:10.

(5) The composite biomaterials according to (4), wherein the ratio ofhydroxyapatite with collagen is between 70:30 and 85:15.

(6) The composite biomaterials according to any one of (1) to (5), inwhich the alginate is homogenously distributed therein.

(7) The composite biomaterials according to (6), wherein the compositebiomaterials after lyophilization have the porosity of 80% or higher.

(8) The composite biomaterials according to (7), which is produced bythe following steps:

-   -   1) mixing a composite of hydroxyapatite and collagen with        alginate; and    -   2) mixing a calcium carbonate suspension with the resulting        mixture, mixing gluconic acid powders thereto to cure the        mixture, and allowing carbon dioxide to foam, thereby obtaining        composite biomaterials.

(9) A process for producing composite biomaterials comprising thefollowing steps:

-   -   1) mixing a composite of hydroxyapatite and collagen with        alginate; and    -   2) mixing a calcium carbonate suspension with the resulting        mixture, mixing gluconic acid powders thereto to cure the        mixture, and allowing carbon dioxide to foam, thereby obtaining        composite biomaterials.

(10) The process according to (9), wherein the composite ofhydroxyapatite and collagen has a microporous structure in which thec-axis of hydroxyapatite is oriented along the collagen fibers.

The present invention is hereafter described in detail.

1. Composite Biomaterials

1.1 Structure of Composite Biomaterials

The composite biomaterials of the present invention compriseshydroxyapatite, collagen, and alginate, and at least a part thereof is amicroporous structure in which the c-axis of hydroxyapatite is orientedalong the collagen fibers. This structure is specific to natural bones,which imparts mechanical properties specific to the compositebiomaterials of the present invention.

The term “microporous structure” used herein refers to a structuresimilar to that of natural bones in which indefinite numbers of pores(gaps) of approximately several μm to several tens of μm are present.

The ratio of hydroxyapatite with collagen in the composite biomaterialsof the present invention is generally between 60:40 and 90:10, andpreferably between 70:30 and 85:15. This is because the ratio thereofneeds to approximate the composition of natural bones (75:25).

The composite biomaterials of the present invention comprise alginateshomogenously distributed therein. This makes the composite biomaterialsmore valuable for applications as implants and the like.

1.2 Constituents of Composite Biomaterials

The alginate content in the composite biomaterials of the presentinvention (after lyophilization) is 1 to 30% by mass, and preferably 5to 20% by mass (“% by mass” is hereafter simply referred to as “%”),relative to the total amount of hydroxyapatite and collagen (totalmass). Specifically, when the amount of alginate is too small, thestrength of the composite becomes insufficient. In contrast, cellinvasion into the biomaterials is blocked when the amount thereof is toolarge.

When the composite biomaterials of the present invention are used asbone fillers without curing and lyophilization, the compositebiomaterials comprise an adequate amount of water, and their watercontents can be adequately determined depending on applications.

When the composite biomaterials of the present invention are used aftercuring and lyophilization, a hydroxyapatite content of 55 to 80%, acollagen content of 10 to 35%, and an alginate content of 1 to 25%,relative to the entire composite biomaterials after lyophilization arepreferable.

The composite biomaterials of the present invention have porosities(foamed portions) of 5 to 70%, and preferably approximately 10 to 50%,in a water-containing state before lyophilization. After thelyophilization, the composite biomaterials have porosities of at least80%, and preferably at least 95%. As mentioned above, low porosityresults in insufficient cell invasion into the biomaterials afterimplantation to the body, which in turn decreases bone inductivity andstrength of the implants.

The lyophilized composite biomaterials of the present invention havegaps (pores) of between 1 μm and 500 μm (average diameter) andindefinite numbers of microgaps (micropores) of 1 μm or smaller. Thismicroporous structure improves cell invasion and bone inductivity afterimplantation to the body.

2. A Process for Producing Composite Biomaterials

The process for producing composite biomaterials of the presentinvention comprises the following steps 1) and 2). Compositebiomaterials having microporous structures in which alginates arehomogenously distributed in composites of hydroxyapatite and collagen(hereafter referred to as “HAp/Col composite”) can be obtained by thisprocess:

1) mixing alginate in a HAp/Col composite; and

2) mixing a calcium carbonate suspension with the resulting mixture,mixing gluconic acid powders thereto to cure the mixture, and allowingcarbon dioxide to foam, thereby obtaining composite biomaterials.

2.1 Step 1

(1) A HAp/Col Composite

A HAp/Col composite used in step 1) preferably has a microporousstructure similar to that of natural bones in which the c-axis ofhydroxyapatite is oriented along the collagen fibers. Such a compositecan be produced in accordance with, for example, the method of Kikuchiet al. (Biomaterials 22, 2000, pp. 1705-1711). More specifically, acomposite of interest can be obtained by simultaneously adding a calciumhydroxide solution and an aqueous phosphate solution containing collagendropwise to a reaction vessel, and dehydrating the resulting sediment.Collagen used herein is not particularly limited. If the molecularweight of collagen is large, however, the strength of a compositebecomes insufficient because of steric hindrance. Accordingly, the useof monomeric collagen is preferable. Pepsin-treated atelocollagen isparticularly preferable for the composite biomaterials of the presentinvention because of its monomeric property and low antigenicity.

Preferably, a small amount of physiological saline, deionized water, aphosphate buffer, or the like is initially added to the above mentionedHAp/Col composite, and the resulting mixture is mixed by a homogenizeror other means. More specifically, when free calcium ion exists in theHAp/Col composite, it is sometimes reacted with alginic acid and causegelatinization. Thus, calcium ion is allowed to adsorb on hydroxyapatiteby adding physiological saline or the like, and it needs to avoidreaction with alginic acid.

When physiological saline or a phosphate buffer is added, ion penetratesthe composite, and it is adsorbed on the surface of hydroxyapatite toneutralize its electric charge. This allows homogenous mixing ofalginate and HAp/Col composite. Thus, the use of physiological saline ora phosphate buffer is particularly preferable. The amount ofphysiological saline, or the like, to be added varies depending on thestructure and composition of the HAp/Col composite. It is preferablybetween 2 times and 6 times the total amount of the HAp/Col composite.

(2) Alginates

Alginates used in step 1) are not particularly limited, and sodium salt,potassium salt, and the like can be used. Crosslinked alginate may beused as alginates. Some of the crosslinked alginate has excellentbioabsorbability, and use thereof is more preferable. Alginates can behandled easily if they are prepared as 3-5% aqueous solutions.

2.2 Step 2

In step 2), gluconic acid and calcium carbonate are neutralized, carbondioxide is then foamed, and alginic acid is cured by being crosslinkedwith calcium ion. Thus, composite biomaterials having microporousstructures can be obtained.

(1) Neutralization (Foaming, Crosslinking)

Calcium carbonate used in step 2) is not particularly limited, and itmay be a suspension or powder. Also, gluconic acid used in step 2) isnot particularly limited.

The molar ratio of calcium carbonate to gluconic acid is between 1:3 and2:3, and preferably about 1:2. The composite biomaterials of the presentinvention can have desired pore sizes and porosities through regulationof the amount of foaming by adequately adjusting the amounts of calciumcarbonate and gluconic acid. When the amounts of calcium carbonate andgluconic acid are too small, gelatinization becomes insufficient. Incontrast, an excess amount thereof results in excessive foaming. Toomuch or too little amount thereof decreases the strength of thecomposite biomaterials. Accordingly, calcium carbonate is preferablyadded in an amount of approximately 10% to 100% relative to the totalamount of the HAp/Col composite. When only gluconic acid is added in anamount larger than the adequate level, the amount of foaming does notvary, while the crosslinking density is elevated by free calcium ionsgenerated from the partially dissolved hydroxyapatite. Thus, thestrength of the implant is enhanced.

When the composite biomaterials of the present invention are intended tobe reinforced, the gelatinized mixture obtained in step 2) is immersedin a calcium hydrochloride solution or the like to crosslink alginicacid. Attention should be given to the density of crosslinking sincecell invasion after implantation to the body will be adversely affectedif crosslinking is too dense.

(2) Curing and Forming

The gelatinized mixture obtained in step 2) begins to cure within aboutseveral minutes to several tens of minutes, and the compositebiomaterials of the present invention can be thus obtained.

The composite biomaterials can be used as bone fillers in that state ifthey are directly implanted to bone defects before curing.

When the production of an implants having a specific configuration andshape is intended, the composite biomaterials are injected into adesired mold immediately after the mixing in of gluconic acid, and thenmolded. When an enhanced level of curing is intended, lyophilization iscarried out. The structures of the composite biomaterials, i.e.,specific surface areas, porosities, pore (gap) sizes, and the like, canbe suitably adjusted by selecting conditions for lyophilization (e.g.,temperature, the duration of freezing, or lyophilization in water).

(3) Others

The composite biomaterials of the present invention may contain theessential components, i.e., hydroxyapatite, collagen, and alginate, aswell as other components within the scope of the present invention.Examples of such components include Bone Morphogenetic Proteins, such asBMP2, BMP6, and BMP7, and growth factors, such as bFGF, aFGF, VEGF, andTGFβ.

3. Applications of Composite Biomaterials

(1) Materials for Bone Regeneration (Implants)

As mentioned above, the composite biomaterials of the present inventioncan be used as bone fillers as they are if they are directly implantedto bone defects before curing. An implant having a desired configurationand shape can be produced by injecting the composite biomaterials into adesired mold immediately after the mixing in of gluconic acid.

The composite biomaterials of the present invention become as elastic assponges and have excellent bioadaptability, bone inductivity, or boneconductivity upon moisture absorption. Specifically, when thebiomaterials are implanted in bone tissues, they rapidly fused with thebone tissues, and integrated into the hard tissue of the recipient.

(2) Scaffold for Dell Culture

The composite biomaterials of the present invention can be used as ascaffold for cell and/or tissue culture. For example, bone marrow,liver, and other tissues can be reconstructed by conducting tissueculture using the composite biomaterials of the present inventioncontaining highly bioactive cytokines as a scaffold under the biomimeticenvironment to which dynamics or electricity had been applied. Suchscaffold enables effective reconstruction of damaged tissues when theyare directly implanted in the body.

(3) Drug Carriers for Sustained Release

The composite biomaterials of the present invention can be used assustained release agents for other bioactive substances, drugs, and thelike. For example, when the composite materials of the present inventionimpregnated with anti-cancer agents are used for reconstructing bonesresected due to osteogenic sarcoma, carcinoma recurrence can beprevented and the generation of hard tissue in the organism can beinduced.

Accordingly, the composite materials of the present invention can beutilized as, for example, materials for bone regeneration capable ofinducing and conducting bones, scaffold for bioactive agents or cellculture in tissue engineering containing amino acids, saccharides, andcytokines, and biocompatible drug carriers for sustained release.Specific examples of applications include artificial bones, artificialjoints, cements for tendons and bones, dental implants, percutaneousterminals for catheters, drug carriers for sustained release, chambersfor bone marrow induction, and chambers or scaffolds for tissuereconstruction.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 is a scanning microscope photograph of the implant prepared inExample 1.

FIG. 2 is a photograph showing an image of HE-stained tissues 2, 4, and8 weeks after the implantation of the implant of the present inventionin Example 2.

FIG. 3 is a photograph showing an image of HE-stained tissues 2, 4, and8 weeks after the implantation of commercialized porous HAp in Example2.

FIG. 4 is a photograph showing an image of HE-stained tissues 2, 4, and8 weeks after the implantation of a HAp/Col composite in Example 2.

FIG. 5 is a photograph showing an image of HE-stained tissues 2, 4, and8 weeks after the implantation of sodium alginate in Example 2.

This description includes part or all of the contents as disclosed inthe description of Japanese Patent Application No. 2001-328167, which isa priority document of the present application.

BEST MODES FOR CARRYING OUT THE INVENTION

The present invention is hereafter described in more detail withreference to the examples, although the technical scope of the presentinvention is not limited thereto.

EXAMPLE 1 Production of Composite Biomaterials ofHydroxyapatite/Collagen/Alginate

1. Method

At the outset, 3 ml of physiological saline was added to 500 mg ofHAp/Col composite powders, the resultant was mixed by a homogenizeruntil a homogenous mixture was obtained. An aqueous solution of 3%sodium alginate (1.5 ml) was then added thereto, and the resultant wasfurther mixed until it became homogenous. HAp/Col composite powders (500mg) synthesized by the method of Kikuchi et al (Biomaterials 22, 2000,pp. 1705-1711) were used as composites of hydroxyapatite and collagen(HAp/Col composites).

Subsequently, 80 μl of a 5M calcium carbonate suspension was addedthereto, the resultant was mixed, and 100 mg of gluconic acid powder wasthen added thereto, followed by mixing.

The resulting mixture was immediately placed in the mold and thenallowed to cure over the period of 45 minutes. This cured product wasallowed to freeze at −20° C. for 12 hours and then lyophilized. Thus,the composite biomaterials (implants) of the present invention wereobtained.

2. Results (Properties of Composite Biomaterials)

Profile sections of the implants were observed using a scanningmicroscope. The implants were found to be porous bodies having pores ofseveral μm to several hundreds of μm (diameter) and had microporousstructures similar to those of natural bones (FIG. 1).

EXAMPLE 2 Experiment of Implantation into Rat Femurs

1. Method

A hole was provided in the distal part of a 10-week-old Wistar ratfemur, and the implants prepared in Example 1 (2×2×5 mm) were implantedthereinto. The implants were taken out 2, 4, 6, and 8 weeks after theimplantation and subjected to HE staining and toluidine blue staining.As controls, the following three kinds of substances were implanted tothe distal part of the rat femurs and evaluated in the same manner asmentioned above. FIGS. 2 to 5 show the results of HE staining 2, 4, and8 weeks after implantation of each sample.

(i) Commercialized sintered porous hydroxyapatite (2×2×5 mm, BONFIL,Mitsubishi Materials Corporation).

(ii) A compressed form of HAp/Col composite used in Example 1 (2×2×5mm).

(iii) A solution of 3% sodium alginate powders (sodium alginate (500-600cP), Wako Pure Chemical Industries, Ltd.) (physiological saline).

2. Results

1) The Implants of the Present Invention

As is apparent from FIG. 2, bones in contact with the implants werealready actively formed in the vicinity of the implants of the presentinvention by the second week. After 4 weeks or 8 weeks, cell invasionalso increased. As is apparent from an enlarged view, in addition to theenhanced cell invasion after 4 weeks or 8 weeks, osteogenesis was alsoenhanced inside the implants. Thus, cell invasion into the implants wasrelatively good, and multinucleated giant cells considered to be“phagocytes” also invaded into the implants. Osteogenesis occurred atthe sites where bones were in direct contact with the implants, andthus, the boundaries between the implants and new bones were unclear. Noinflammatory reaction was observed as a result of toluidine bluestaining.

2) Commercialized Sintered Porous Hydroxyapatite

As is apparent from FIG. 3, bones in contact with the hydroxyapatitewere already actively formed in the vicinity of the commercializedhydroxyapatite by the second week. However, cell invasion into thehydroxyapatite did not increased very much even after 4 weeks or 8weeks. As is apparent from an enlarged view, osteogenesis was alsoenhanced inside the hydroxyapatite after 4 weeks or 8 weeks. Also, inspite of its porous structure, cell invasion was not found in most ofthe pores. Intensive inflammatory reaction was not observed.

3) A Compressed form of HAp/Col Composite

As is apparent from FIG. 4, cell invasion into the HAp/Col composite wasnot found by the second week. Although resorption of the HAp/Colcomposite made progress after 4 weeks or 8 weeks, cell invasion did notyet increased into the HAp/Col composite that were not resorbed. As isapparent from the enlarged view, resorption of HAp/Col composite madeprogress with time, and cell invasion was found at the site of theresorption. Osteogenesis took place outside of the fibrous tissue insuch a manner that osteogenesis followed the implant resorption.

4) A Solution of 3% Sodium Alginate

As is apparent from FIG. 5, bones partially in contact with the sodiumalginate were formed. However, the degree of cell invasion was low evenafter 8 weeks. Intensive inflammatory reaction was not observed. Anenlarged view also represents the similar results.

3. Conclusions

Accordingly, the implants (composite biomaterials) of the presentinvention were found to have bioadaptability, the capacity for cellinvasion, and the capacity for osteogenesis better than other poroussubstances. The composite biomaterials of the present invention werefound to be excellent in terms of safety since no inflammatory reactionwas observed after implantation.

All publications, patents, and patent applications cited herein areincorporated herein by reference in their entirety.

INDUSTRIAL APPLICABILITY

The present invention provides novel composite biomaterials havingexcellent bioadaptability and bone inductivity.

1. Composite biomaterials, which comprise hydroxyapatite, collagen, andalginate and have a microporous structure in which alginates arehomogenously incorporated therein and the c-axis of hydroxyapatite isoriented along the collagen fibers, wherein the alginate is homogenouslydistributed therein.
 2. The composite biomaterials according to claim 1,wherein an alginate content is 1 to 30% by mass, relative to the totalamount of hydroxyapatite and collagen.
 3. The composite biomaterialsaccording to claim 2, wherein an alginate content is 5 to 20% by mass,relative to the total amount of hydroxyapatite and collagen.
 4. Thecomposite biomaterials according to claim 3, wherein the ratio ofhydroxyapatite with collagen is between 60:40 and 90:10.
 5. Thecomposite biomaterials according to claim 4, wherein the ratio ofhydroxyapatite with collagen is between 70:30 and 85:15.
 6. Thecomposite biomaterials according to claim 1, wherein the compositebiomaterials after lyophilization have the porosity of 80% or higher. 7.The composite biomaterials according to claim 6, which is produced bythe following steps: 1) mixing a composite of hydroxyapatite andcollagen with alginate; and 2) mixing a calcium carbonate suspensionwith the resulting mixture, mixing gluconic acid powders thereto to curethe mixture, and allowing carbon dioxide to foam, thereby obtainingcomposite biomaterials.
 8. A process for producing compositebiomaterials comprising the following steps: 1) mixing a composite ofhydroxyapatite and collagen with alginate; and 2) mixing a calciumcarbonate suspension with the resulting mixture, mixing gluconic acidpowders thereto to cure the mixture, and allowing carbon dioxide tofoam, thereby obtaining composite biomaterials.
 9. The process accordingto claim 8, wherein the composite of hydroxyapatite and collagen has amicroporous structure in which the c-axis of hydroxyapatite is orientedalong the collagen fibers.
 10. (canceled)